[Histonet] My Negative is now Positive ... IHC assistance needed?

[GT Hebert]

Jan,
 
How do you know the initial concentration of IgG in the Normal Rabbit Serum (is it given to you by DAKO?)?  Sounds like a good switch if I actually can dilute the serum IgG to the same concentration as my primary.  Let me know.  Thanks.

Jan Shivers <shive003 <@t> umn.edu> wrote:
Try DAKO's Normal Rabbit Serum (X0902). Works nicely for me as negative
control to accompany my rabbit polyclonals, when diluted to the same Ig
concentration as the primary is used at.

Jan Shivers
U of MN Vet Diag Lab

----- Original Message ----- 
From: "GT Hebert" 
To: 
Sent: Thursday, June 02, 2005 4:14 PM
Subject: [Histonet] My Negative is now Positive ... IHC assistance needed?


> Hello,
>
> I recently worked up a new antibody for my lab. It is a rabbit
> anti-mouse (antigen). The antigen is found throughout the kidney and has
been
> pinpointed by ISH to be located in the proximal tubules. For the IHC,
> I have determined the best concentration for my antibody was 7 ug/ml.
> I used Jackson's ChromPure Rabbit IgG, whole molecule as a negative
> control at the same concentration, diluted in the same buffer as my
> primary. I am also using the Envision system by DAKO.
>
> Lately I've noticed a lot of background coming up with my negative. I
> re-ran a titre for my primary and had negatives at the corresponding
> concentrations. To my surprise, I got a lot of background on the
> negative, and moreover, it appears to be decreasing as the dilutions get
> higher.
>
> OK -- what's going on? Have I contaminated my negative somehow?? I
> keep the glass jar it originally came in at 4 degree C. When needed, I
> aliquot what I need under the Biosafety Cabinet (sterile conditions).
> If it has been contaminated, what has it been contaminated with and why
> is it giving me background? How can I avoid this in the future (can it
> be aliquoted and frozen)? Other suggestions will be very helpful.
>
> My positive looks good. Very clear staining and background only seen
> when Ab concentration is very high. I need a clear negative though to
> support my primary Ab results.
>
> Thank you all again for your help.
>
>
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